RecA+ protein (wild type) is a 38,000 dalton protein found in the bacterium Escherichia coli, which is important for homologous DNA recombination. Most information about its biochemistry and enzymology comes from studies on purified RecA+ protein. Numerous in vitro studies have shown that RecA+ protein is intimately involved in the pairing reaction between homologous DNA sequences that ultimately leads to homologous recombination events (see Cox et. al. for a recent review of RecA+ protein properties). It is this pairing reaction that makes RecA+ protein highly useful for DNA diagnostics and therapeutics applications.
In the presence of ATP, RecA+ protein catalyzes strand exchange between a number of substrates, the most relevant for DNA probe applications being single- and double-stranded DNAs. Single-stranded DNA (probe) interacts with the homologous portion of the double-stranded ("native") target sequences, initially by forming a recombination intermediate containing hybridized, partially joined molecules. This is followed by branch migration, and forming of fully hybrid molecules between the original single- and double-stranded DNAs, depending upon the extent of their homology. This reaction results in a product that is a hybrid between probe and target. Such hybrids can be easily detected using, for example, radio-labeled, enzyme-labeled, chemiluminescently-labeled, phosphorescently-labeled or fluorescently-labeled probes.
The present application demonstrates the feasibility of using RecA+ protein to facilitate and improve the efficiency of hybridization reactions involving single-stranded primer and complementary native double-stranded target sequences. In particular, RecA+ protein is particularly well suited for many DNA probe applications because the double-stranded target DNA does not need to be denatured (e.g., by heating) before hybridization. Further, RecA+ protein is useful in facilitating the initiation and completion of DNA chain elongation at DNA sequences that are either damaged or difficult to denature.